Gel electrophoresis: sort and see the DNA
Pre-class activity
Directions:
:
1.
Go to the DNAi website www.dnai.org > Manipulation > Techniques > sorting and sequencing.
2. View the Gel Electrophoresis 2-D animation, and answer the following questions.
Questions:
1.
How does the process of gel electrophoresis separate DNA fragments?
Using electric current and a gel as a filter, fragments are
separated based on size.
2. What is the purpose of the agarose gel?
It acts as a filter and the higher the % gel (i.e. 1% vs. 5%)
the more finely the filter acts - fragments that are close in size
can be separated better with a higher concentrated gel.
3. What is the purpose of adding blue “tracking” dye to the DNA samples?
DNA is invisible to naked eye in the gel and the tracking dye is
known to move faster than smallest fragment so it allows you to
determine when the DNA has moved to the opposite end of the gel.
4. Explain why DNA has an overall negative charge.
DNA is surrounded by a sugar phosphate backbone (both sides of the
ladder) and phosphate is negatively charged. So overall charge of
DNA is negative.
5. Why is the fact that DNA has a negative charge so important in the gel electrophoresis process?
Because it is negative, it can be moved through an electric field
and fragments move toward the positive terminal.
6. Explain how an agarose gel can separate DNA fragments of different lengths.
Fragments are being pulled through a medium (gel), the shorter
fragments are easier to pull through than are the larger.
7. What is the purpose of ethidium bromide in gel electrophoresis?
After the gel runs, it is applied and will bind to the DNA so that
bands are visible with UV light.
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8. Why is a marker used when running the fragments through the gel?
The marker contains fragments of known size so they run with your
sample and then can be used to compare your sample with a kind of
ruler - must always run with each sample to match the conditions.
9. What is a restriction map?
A diagram (map) based on the length of fragments created with
specific restriction enzymes - that can be used to identify where
those restriction enzyme sites are.
10.
On the gel picture below,
(a) circle the smallest fragment produced by a restriction enzyme and label it “smallest.”
(b) circle the largest fragment produced by a restriction enzyme and label it “largest.”
11. In one or two sentences, summarize the technique of gel electrophoresis.
Gel electrophoresis separates DNA molecules based on size
using an electric current and agarose which defines largest
or smallest molecules based on movement. Dye is used to
stain the bands in order to view them and loading dye is used
to monitor the progress of the DNA movement.
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Restriction maps of the linear l genome
Lambda (l)
0
10,000
20,000
30,000
40,000
48,502
l----------------------l-------------------------l-------------------------l--------------------------l-------------------------l
EcoRI Sites
21,226
26,104
31,747
39,168
44,972
----------------------------------------------------l------------l--------------l-------------------l------------------l--------
HindIII Sites
25,157
23,130
37,459
27,479
36,895
37,584
44,141
---------------------------------------------------------l-----------l----------------------l-ll---------------------l----------
BamHI Sites
5,505
22,346
27,972
34,499
41,732
-------------l-----------------------------------------l--------------l-----------------l------------------l--------------------
NcoI Sites
19,329
23,901 27,868
44,238
-----------------------------------------------l------------l---------l------------------------------------------l--------------
BmrI Sites
7,054
11,608
25,691
30,332
----------------l-----------l-----------------------------------l-----------l---------------------------------------------------
StuI Sites
32,997
12,434
31,478
40,596
39,992
40,614
------------------------------l------------------------------------------------l-----------------------l---l-l--------------------
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Gel electrophoresis: sort and see the DNA
Making a DNA fingerprint
In this activity, you will model the construction of DNA fingerprints for a viral genome using different
restriction enzymes. You will also practice interpreting restriction maps and visualize how the process of gel
electrophoresis separates DNA fragments.
DNA restriction fragment size chart
Directions:
List your DNA fragments in the following chart under the column of the appropriate restriction enzyme.
List each fragment, from largest to smallest.
EcoRI
HindIII
BamHI
NcoI
BmrI
StuI
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DNA fingerprints
Marker
EcoRI
HindIII
BamHI
NcoI
BmrI
StuI
(50,000)
(30,000)
(20,000)
(15,000)
(10,000)
(5,000)
(2,500)
(1,000)
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